Proteins are major players carrying out vital functions in the body, and in order to systematically exert their functions in the cellular society, post-translational modifications such as sugar chain modifications play a very important role. Nearly all proteins in the body undergo sugar chain modifications. Recently, there has been a string of reports revealing that sugar chains added to proteins play important roles in various life phenomena such as viral infections, protozoan parasitism and infections, toxin binding, hormone binding, fertilization, development and differentiation, protein stability, cancer cell metastasis and apoptosis.
To analyze the function of a sugar chain, it is first essential to analyze the sugar chain's structure. The importance of methods for analyzing sugar chain structure is predicted to increase in the future. However, since analyses of sugar chain structures require considerable time, labor, and experience, instead of aiming to completely determine structures based on conventional techniques, the development of systems capable of extracting the characteristics of a diversity of sugar chain structures, and mutually distinguishing these structures with greater ease, speed, sensitivity and accuracy, has been expected.
Microarray is a generic term for an apparatus onto which various types of immobilized samples, such as DNAs and proteins, are immobilized on a solid phase carrier (glass, membrane, or silicon chip) in the form of high density spots; microarrays can detect the presence or absence of molecules (hereinafter referred to as probes) that specifically bind to the various sample spots immobilized onto the carrier. The probe molecules used are typically fluorescently labeled, and after reacting a probe solution with an array surface, probe molecules that have bound to each sample spot can be quantitatively analyzed by observation using a fluorescence detection scanner. Since the development of a DNA microarray by Affimetrix Corp. in the U.S., microarrays have been used in an extremely wide range of research fields and have brought various new findings to the human race.
If, when studying the structural and functional information of sugar chains, which are called the third life chain, it were possible to use a microarray for the rapid and highly sensitive large-scale analysis of the interactions between sugar chains and the proteins that interact with sugar chains (sugar-binding proteins, for example lectins, etc), then this could conceivably become an extremely useful tool, applicable over a wide range of applications, from basic research to medical diagnoses and industrial applications.
Compared to the typical dissociation constants and such of antigen-antibody reactions (Kd=10−8 or less), the binding between sugar chains and proteins that interact with sugar chains is known to generally be a weak interaction, with dissociation constants (Kd) frequently 10−6 M or more. In addition, the interactions between sugar chains and proteins that interact with sugar chains are known to consist of relatively rapid dissociation-association reactions. As a result, the equilibrium tends to shift towards dissociation due to washing procedures and such, as compared to typical protein-protein interactions or interactions between complementary nucleotide fragments. For example, when purifying lectins with a glycoprotein-immobilized column and such, the lectins are frequently observed to run off the column during the washing procedure, when their binding is weak.
In typical microarray technology using a conventional slide glass, a probe solution is contacted with an immobilized sample and a binding reaction takes place, then the probe solution is washed away, and moisture adhering to the slide glass is completely removed using a jet of gas or a centrifuge, followed by imaging using a microarray scanner. This is because a typical microarray reader cannot examine fluorescence on a slide glass on which there is moisture adhered. Since the dissociation rate constant is sufficiently small for strong binding interactions, such as those between complementary nucleotide fragments and antigen-antibody reactions, the dissociation reaction of probe molecules is not thought to proceed easily, even when the probe solution is removed at a stage prior to scanning. However, when examining interactions with a large dissociation constant, i.e. the weak interactions generally seen between sugar chains and proteins that interact with sugar chains, a dissociation reaction proceeds between these sugar chains and proteins upon removal of the probe solution and the washing procedure, making it difficult to obtain accurate data on interactions under conditions of equilibrium. Consequently, this procedure of washing the probe solution presents a significant problem when accurately analyzing data on the interactions between sugar chains and proteins that interact with sugar chains under conditions of equilibrium in a microarray.
DNA microarrays are currently in a wide range of use. Future application of protein microarrays is expected in basic research fields involving the elucidation and such of the functions of proteins, which are the transcription products of DNA, in the body, and in application fields involving diagnoses, evaluation, and such based on quantitative and qualitative protein changes. Active studies are also being conducted throughout the world in the field of research. However, the development and popularization of protein microarrays is currently far behind that of DNA microarrays. One of the reasons for this, as pointed out early on by numerous researchers, is that it is technically very difficult to immobilize protein samples with various differing properties at a constant rate, while maintaining their activity.
Examples of methods for immobilizing proteins on an array comprise a method developed very early on, in which proteins are physically adsorbed onto a membrane, as exemplified by PVDF membranes (Non-Patent Document 1). Although there are reports that activity is maintained to a certain extent for some proteins such as transcription factors, this is generally not the case. In addition, array density was limited when immobilizing proteins onto a membrane. Although research has progressed towards the immobilization of proteins onto solid surfaces such as metal and glass to achieve higher densities, proteins are generally easily denatured by contact with a solid surface such as metal or glass. Consequently, dedicated research and development have been conducted on immobilization methods that use some linker to crosslink the solid surfaces and proteins.
An example of a method for reducing the problem of protein denaturation involves a method in which a polyacrylamide pad 10 μm to 100 μm thick is attached onto a slide glass, followed by the spotting of proteins (Non-Patent Documents 2 and 3). In this case, since the proteins are immobilized in a three-dimensional space, a quantitative improvement of 100 times or more can be expected compared to methods of immobilization onto a two-dimensional surface. In addition, there is also a method in which proteins are immobilized in a porous polyacrylamide gel via their amino groups (Non-Patent Document 4). However, these methods have not been popularized since they are costly and require the production of special slide glasses. In addition, depending on the detection method, a thick layer of immobilized proteins may not be preferable.
One method for immobilizing proteins onto a solid phase, which is now being most actively investigated, is a method by which proteins are expressed with some tag attached thereto, and this tag is used to immobilize the protein onto a solid carrier. This method is said to improve the effective ligand concentration of the proteins, and to theoretically allow alignment of protein orientation. Examples of such methods comprise a method for using oligohistidine tags to immobilize proteins onto a substrate whose surface is modified with a nickel complex (Non-Patent Document 5), and a method for immobilizing via avidin-biotin (Patent Document 1).
These methods are considered to be effective in terms of immobilizing proteins while retaining their activity or enabling a uniform immobilization rate. However, it is expensive and labor-intensive to add a tag at the genetic level to all proteins for which immobilization onto a microarray is being attempted, and to then express these proteins in Escherichia coli, a cell-free system or such, and purify them. Thus, at the present time, these methods are difficult for ordinary researchers to use easily and in a form that flexibly responds to individual needs.
In contrast, methods that utilize protein functional groups to immobilize proteins onto a solid phase carrier can characteristically immobilize proteins extracted from nature as is, or commercially available protein samples as is, for use in microarrays. Examples of methods for immobilizing proteins onto a solid phase carrier via protein amino groups comprise methods in which proteins are immobilized via active ester groups bound to the solid phase surface, and methods in which proteins are immobilized via epoxy groups arranged on the solid phase surface (Non-Patent Document 6). Methods for immobilizing proteins via their amino groups are simple, however, they also enable easy immobilization of commercially available proteins, biological extracts and components, recombinant proteins without specific tags, and such. Therefore, individual users are able to freely select a protein that suits their purpose, and to rapidly and inexpensively optimize this protein for use in a microarray that suits the purpose. Examples of disadvantages in the methods in which proteins are immobilized via amino groups include the fact that the number of lysine residues in a protein differs for each protein, and there is a possibility of inactivating the protein depending on the location of the lysine group used for immobilization.    [Patent Document 1] Japanese Patent Application No. 2001-520104    [Patent Document 2] Japanese Patent Application Kokai Publication No. (JP-A) H08-201383 (unexamined, published Japanese patent application)    [Patent Document 3] Japanese Patent Kohyo Publication No. (JP-A) 2002-544485 (unexamined    Japanese national phase publication corresponding to a non-Japanese international publication)    [Non-Patent Document 1] L. J. Holt, K. Bussow, G. Walter, I. M. Tomlinson, Nucleic Acids Res., 15, E72, 2000    [Non-Patent Document 2] D. Guschin, G Yershov, A. Zaslaysky, A. Gemmell, V. Shick, D. Proudnikov, P. Arenkov, A. Mirzabekov, Anal. Biochem., 250, 203-211, 1997    [Non-Patent Document 3] A. Lueking, M. Horn, H. Eickhoff, K. Bussow, H. Lehrach, G Walter, Anal. Biochem., 270, 103-111, 1999    [Non-Patent Document 4] P. Mitchell, Nat. Biotechnol., 20, 225-229, 2002    [Non-Patent Document 5] H. Zhu, M. Bilgin, R. Bangham, D. Hall, A. Casamayor, P. Bertone, N. Lan, R. Jansen, S. Bidlingmaier, T. Houfek, T. Mitchell, P. Miller, R. A. Dean, M. Gerstein, M. Snyder, Science, 293, 2101-2105, 2001    [Non-Patent Document 6] H. Zhu, J. F. Klemic, S. Chang, P. Bertone, A. Casamayor, K. G. Klemic, D. Smith, M. Gerstein, M. A. Reed, M. Snyder, Nat. Genetics. 26, 283-289, 2000